WORLD HEALTH ORGANIZATION
WHO Food Additives Series 1972, No. 1
TOXICOLOGICAL EVALUATION OF SOME
ENZYMES, MODIFIED STARCHES AND
CERTAIN OTHER SUBSTANCES
The evaluations contained in this publication were prepared by the
Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
16-24 June 19711
World Health Organization
1 Fifteenth Report of the Joint FAO/WHO Expert Committee on Food
Additives, Wld Hlth Org. techn. Rep. Ser., 1972, No. 488; FAO
Nutrition Meetings Report Series, 1972, No. 50.
The monographs contained in the present volume are also issued by the
Food and Agriculture Organization of the United Nations, Rome, as FAO
Nutrition Meetings Report Series, No. 50A
(c) FAO and WHO 1972
Modification is carried out by propylene oxide at levels up to 25 per
cent. and the resultant starch is usually lightly oxidized, bleached
or acid modified after etherification. Substitution may amount to a
maximum of 40 ether linkages per 100 glucopyranose units if 25 per
cent. propylene oxide is used, and 4-6 ether linkages per 100
glucopyranose units if 5 per cent. propylene oxide is used.
In vitro digestibility by pancreatin was estimated by comparing
the amount of reducing material liberated with that formed from native
wheat starch. No significant difference could be detected between low
(1 in 10) and high (4 in 10) substituted starches compared with
unmodified starch (Kay & Calandra, 1962). In contrast the
digestibility by pancreatin was found to decrease with increased
substitution degree. At 0.04 degree the digestibility was 80 per
cent. of that of unmodified starch (Leegwater & Luten, 1971). Corn
starch treated with propylene oxide -2-C14 to produce
hydroxypropyl starch (degree of substitution 0.12) was given to a male
rat by gavage. Ninety-two per cent. of the radioactivity was excreted
in the faeces and 3.6 per cent. in the urine over the next 50 hours.
The urinary activity was probably derived from propyleneglycol in the
test material. Further investigation of the degradation products in
the faeces revealed hydroxypropyl maltose to be the major metabolite
in vitro and in vivo (Leegwater, 1971).
Groups of 10 male and 10 female rats were fed for 90 days diets
containing 0, 2, 5, 10 and 25 per cent. of highly modified starch (25
per cent. propylene oxide) and 25 per cent. unmodified starch. No
systemic toxicity was noted. There were no adverse effects regarding
mortality, urinalysis or haematology at any level. There was slight
reduction in growth rate at the highest dietary level with lower food
utilization and without an equivalent increase in food consumption.
Mild diarrhoea occurred at 25 per cent. dietary level. No adverse
effects occurred at any other level. At autopsy there were no
significant differences in the organ weights of liver, kidney, spleen,
gonad, heart or brain. The observed increased ratios at 25 per cent.
dietary level for liver/body-weight and kidney/body-weight were due to
the relatively lower body-weight. Gross and histological examination
of all major tissues revealed no abnormalities due to the feeding of
highly modified starch (Kay & Calandra, 1961). In another experiment
groups of 10 male and 10 female rats were fed for 90 days on diets
containing 0, 5, 15 and 45 per cent. of low modified starch (5 per
cent. propylene oxide). Haematological findings at 12 weeks were
comparable for all groups. Body-weights did not differ significantly
from controls but were consistently lower in male rats only. Feed
efficiency was similar in all groups. Caecal enlargement was seen at
the 45 per cent. and very slightly at the 15 per cent. level. No
histological abnormalities were detected in any major organs, which
were due to the test substance. The enlarged caeca showed no evidence
of inflammation or changes in the muscular coat (Feron et al., 1967).
Biological data on propylene chlorohydrin
Propylene chlorohydrin was identified as the residue formed in foods
fumigated with propylene oxide (Wesley et al., 1965). The
chlorohydrin is formed by the reaction of the epoxide with the
chloride of food. Previously, it had been believed that propylene
glycol, formed by reaction with water in the food, was the residue.
Propylene chlorohydrin is also formed in starches modified by
hydroxypropylation. Both propylene chlorohydrin isomers have been
identified in fumigated foods (Ragelis et al., 1966).
When volatilization was precluded, a combination of high temperature
and prolonged time in cooking did not appreciably alter the propylene
chlorohydrin content of food, but when volatilization was possible,
the chlorohydrin content was reduced 50 per cent. by cooking (Wesley
et al., 1965). When propylene chlorohydrin was added to a standard
ground laboratory rat diet, 20 minutes of mixing in an open mixer at
room temperature resulted in a 65 per cent. decrease in the propylene
chlorohydrin content (USFDA, 1966).
Animal Route LD50 Reference
rat oral 218 USFDA, 1969
dog oral 150 mg/kg - no deaths USFDA, 1969
200 mg/kg - 1/7 deaths
250, 300 mg/kg - 6/6 deaths
Groups of 10 male and 10 female five-week-old rats were fed, for 25
weeks, diets to which propylene chlorohydrin had been added. The
planned dietary levels were 0, 1000, 2500, 5000 and 10 000 ppm, but
analysis of the 10 000 ppm diet after mixing in the test compound
(open mixer, 20 minute mixing time, room temperature) showed an actual
concentration of 3568 ppm or 35 per cent. of the planned level. The
2-chloro isomer constituted 27 per cent. of the total found. The
actual level in this diet, after 7 days exposure to laboratory
conditions, was reduced to 838 ppm, with 32 per cent. of the 2-chloro
isomer, or less than 10 per cent. of the planned concentration.
Weight gain in both sexes on the 5000 and 10 000 ppm levels was
depressed. The depression was slight in the males on the 5000 ppm
level and both groups of females and moderate in the males on the
10 000 ppm level. Food consumption was slightly decreased in these
groups but food efficiency was normal. The average liver and kidney
weights of the males and the liver weight of the females on the 10 000
ppm level were decreased but the organ weight/body-weight ratios were
normal. The decreased spleen weights and spleen/body-weight ratios in
the males and other minor organ weight variations appeared to be
unrelated to the treatment. No effects on haematological values,
mortality, or gross or microscopic lesions in the tissues were
observed (USFDA, 1969).
Propylene chlorohydrin was administered to groups of 10 male and 10
female eight week old rats by stomach tube in doses of 0, 25, 50, 75
and 100 mg/kg/day for 22 weeks. The dose for the high level was
increased from 100 mg/kg to 150 mg/kg in the eleventh week, to 200
mg/kg in the fourteenth week, and to 250 mg/kg in the sixteenth week.
Doses of 200 mg/kg and less did not increase mortality. All the rats
on the high level were dead by the nineteenth week with all but one of
the deaths occurring between the sixteenth and nineteenth weeks after
the dose had been increased to 250 mg/kg. On the high level, weight
gain was moderately depressed in the males and slightly depressed in
the females while the dose was 100 or 150 mg/kg. Both sexes lost
weight when the dose was increased to 200 mg/kg. Weight gain was
slightly, but not significantly, decreased in both sexes on the 75
mg/kg level. Food consumption was slightly decreased in the males of
the high level while the dose was 100 mg/kg and decreased to a greater
extent when the dose was raised. The females on the high level also
showed a slight decrease in food consumption when the dose was
increased. With the rats losing weight when the dose was increased to
200 mg/kg, the food efficiency values have no meaning. The liver
weight/body-weight ratios of both males and females on the 75 mg/kg
dose and the liver weight and liver weight/body-weight ratio of the
males on the 25 mg/kg dose were increased, but this increase was not
accompanied by gross or microscopic alterations in the liver. Other
organ weight and organ weight/body-weight ratio changes did not appear
to be related to the treatment. No haematological effects or gross or
microscopic effects on the tissues of the treated rats, at a dose of
75 mg/kg or less, were seen. The tissues of the high level rats were
not examined microscopically (USPDA, 1969).
Short-term feeding studies with rats show that even the most highly
modified starch tested is well utilized.
The metabolic study in rats using radio-labelled material shows that
most of the radio-labelled hydroxypropyl containing moiety is excreted
in the faeces. No long-term study on this modified starch is
available but collateral evidence from the long-term study in rats
with hydroxypropyl distarch glycerol, a more high modified starch,
indicates that the propylene glycol moiety is causing no adverse
effects. The available evidence for the group of modified starches
considered suggests that caecal enlargement without associated
histopathological changes is of little toxicological significance.
Temporarily not limited.*
Further work required by 1973
Histological report to complete the two-year rat study on
hydroxypropyl distarch glycerol.
Histological report to complete the reproduction study on
hydroxypropyl distarch glycerol.
Feron, V. J., Til, H. P. & de Groot, A. P. (1961) Unpublished report
No. R 2456 by Centraal Instituut voor Voedingsonderzoek
Kay, J. H. & Calandra, J. C. (1961) Unpublished report by Industrial
Bio-Test Laboratories, Inc.
Kay, J. H. & Calandra, J. C. (1962) Unpublished report by Industrial
Bio-Test Laboratories, Inc.
Leegwater, D. C. (1970) Unpublished report No. R 3209 by Centraal
Instituut voor Voedingsonderzoek
Leegwater, D. C. (1971) Unpublished report No. 3441 by Centraal
Instituut voor Voedingsonderzoek
*Except for good manufacturing practice.
Leegwater, D. C. & Luten, J. B. (1971) Unpublished report No. 3440 by
Centraal Instituut voor Voedingsonderzoek
Majors, P. A. & Ruben König, H. L. (1959) Unpublished report by Hill
Top Research Institute, Inc.
Ragelis, E. P., Fisher, B. S. & Klimeck, B. A. (1966) J.O.A.C., 49,
United States Food and Drug Administration (1969) Unpublished report
Wesley, P., Rourke, B. & Darbishire, O. (1965) J. Fd. Sci., 30, 1037